WO2015044426A1 - Procédé, dispositif et système permettant de commander électrochimiquement dans l'espace la formation d'un hydrogel - Google Patents

Procédé, dispositif et système permettant de commander électrochimiquement dans l'espace la formation d'un hydrogel Download PDF

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Publication number
WO2015044426A1
WO2015044426A1 PCT/EP2014/070820 EP2014070820W WO2015044426A1 WO 2015044426 A1 WO2015044426 A1 WO 2015044426A1 EP 2014070820 W EP2014070820 W EP 2014070820W WO 2015044426 A1 WO2015044426 A1 WO 2015044426A1
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Prior art keywords
solution
moiety
hydrogel
electrode
linking reaction
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PCT/EP2014/070820
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English (en)
Inventor
Vincent MILLERET
Benjamin R. SIMONA
János VÖRÖS
Martin Ehrbar
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Universität Zürich
ETH Zürich
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Priority claimed from EP13186561.0A external-priority patent/EP2853620A1/fr
Application filed by Universität Zürich, ETH Zürich filed Critical Universität Zürich
Priority to ES14790010T priority Critical patent/ES2880311T3/es
Priority to JP2016544768A priority patent/JP6518673B2/ja
Priority to EP14790010.4A priority patent/EP3049554B1/fr
Priority to DK14790010.4T priority patent/DK3049554T3/da
Priority to US15/025,559 priority patent/US10385170B2/en
Publication of WO2015044426A1 publication Critical patent/WO2015044426A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/04—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers only
    • C08G65/06—Cyclic ethers having no atoms other than carbon and hydrogen outside the ring
    • C08G65/08—Saturated oxiranes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/26—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers and other compounds
    • C08G65/2696—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers and other compounds characterised by the process or apparatus used
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
    • C25B11/00—Electrodes; Manufacture thereof not otherwise provided for
    • C25B11/02—Electrodes; Manufacture thereof not otherwise provided for characterised by shape or form
    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
    • C25B11/00—Electrodes; Manufacture thereof not otherwise provided for
    • C25B11/04—Electrodes; Manufacture thereof not otherwise provided for characterised by the material
    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
    • C25B3/00—Electrolytic production of organic compounds
    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
    • C25B3/00—Electrolytic production of organic compounds
    • C25B3/20—Processes
    • C25B3/29—Coupling reactions
    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
    • C25B3/00—Electrolytic production of organic compounds
    • C25B3/20—Processes
    • C25B3/29—Coupling reactions
    • C25B3/295—Coupling reactions hydrodimerisation
    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
    • C25B9/00—Cells or assemblies of cells; Constructional parts of cells; Assemblies of constructional parts, e.g. electrode-diaphragm assemblies; Process-related cell features
    • C25B9/30—Cells comprising movable electrodes, e.g. rotary electrodes; Assemblies of constructional parts thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2210/00—Compositions for preparing hydrogels

Definitions

  • the invention relates to a method, a device and a system for spatially and locally controlling a formation of a chemical link between a first and a second chemical compound in solution, particularly catalyzed by means of an enzymatic reaction and wherein the invention can particularly be used for producing a spatially structured hydrogel.
  • Three-dimensional (3D) engineered tissues are largely desirable to allow physiological studies and the development of regenerative therapies.
  • Most of the recent attempts to build tissue mimetics are based on the culture of cells in extracellular matrix (ECM) hydrogels.
  • ECM extracellular matrix
  • synthetic hydrogels such as poly(ethylene glycol) (PEG) have no intrinsic interaction with biological systems, hence don't trigger - nor are affected by biological processes.
  • PEG hydrogels an ideal platform for the emulation of naturally occurring ECM [1].
  • TG-PEG hydrogels which are polymerized by a Factor XIII (FXIII)-mediated transglutamination (TG) reaction and thus are referred to as TG-PEG hydrogels [2].
  • FXIII Factor XIII
  • TG-PEG hydrogels During the FXIII-mediated crosslinking of the Glutamine-PEG (Gln-PEG) and the Lysine-PEG (Lys-PEG) precursors, various Gln- and Lys-tagged peptides and proteins can be covalently bound to the forming matrix [3, 4].
  • the enzymatic cross-linking reaction allows the site specific, orthogonal integration of bioactive molecules under physiological conditions to provide various functionalities [5, 6].
  • Electrochemical polymerization e.g. oxidative polymerization
  • polyaniline polypyrroles
  • polyacrylates polyacrylates
  • other electroactive polymers for applications spanning from organic electronics to organic film deposition on biomedical implants [16].
  • the problem underlying the present invention is to provide a method, a device and a system allowing the spatial control of formation of a covalent bond particularly for use in a linking reaction for a hydrogel and particularly to produce spatially controlled microenvironments.
  • a method for controlling a linking reaction in a solution in proximity of a first electrode alters the pH of the solution locally for controlling said linking reaction in the proximity of said electrode by applying an electrolysis-inducing electrical current to the solution via said first electrode.
  • a second electrode is provided.
  • the first electrode can assume anodic polarization or cathodic polarization, turning it into an anode or a cathode.
  • a second electrode would assume the opposite polarity of the first electrode.
  • a linking reaction particularly refers to the chemical linking particularly via the formation of a covalent bond between two chemical compounds, particularly a first and a second compound comprising a first and a second moiety between which the link and particularly the covalent bond is established.
  • one or several parameters of the linking process are changed during the linking process (this applies to all embodiments of the present invention).
  • composition of the crosslinking solution e.g. buffer, enzymes, substrate of the enzyme, the used polymers and/or their functional groups
  • the composition of the crosslinking solution e.g. buffer, enzymes, substrate of the enzyme, the used polymers and/or their functional groups
  • a spatially structured hydrogel is formed by said linking reaction, wherein said hydrogel is particularly formed by local inhibition of the linking reaction, wherein said inhibition is particularly controlled by altering said pH locally, particularly such that said altered pH affects the enzymatic activity of an enzyme in the solution, and wherein particularly said enzyme is converting a first and a second moiety comprised by a first and a second compound in the solution to said hydrogel by means of forming a covalent bond between said first and second moiety.
  • a spatially structured hydrogel is particularly a hydrogel that has a consistency that varies in space.
  • Such structures comprise particularly micro-channels or cavities particularly in the ⁇ to mm range.
  • a spatially structured hydrogel comprises locations where despite there was solution present at these locations during formation of the hydrogel and wherein said solution comprised all necessary precursors, as for example the first and second chemical compound comprising the first and second moiety and an enzyme, the formation of the hydrogel was particularly inhibited in these location, such that at these location no hydrogel was formed and thus a spatially structured hydrogel is obtained.
  • spatially structured also refers particularly to the spatially controlled deposition of chemical compounds particularly in and within a hydrogel, particularly for providing a biological microenvironment.
  • the linking reaction is spatially and/or temporally confined by means of adjusting the magnitude and direction of the electrical current flowing through the solution.
  • a method for controlling an enzymatically-catalysed formation of a covalent bond in a solution is claimed, particularly using the method according to the invention described above, wherein said covalent bond is formed between a first compound comprising a first moiety and a second compound comprising a second moiety, wherein the first and the second moiety are a substrate of an enzyme wherein said enzyme catalyzes the formation of a covalent bond between the first and the second moiety, and wherein a voltage is applied to the solution for spatially controlling said formation, wherein said voltage is adjusted such that it induces electrolysis of said solution, wherein said electrolysis induces a spatial pH distribution in said solution, said distribution being aligned particularly along the gradient of the voltage induced electric field, such that the particularly pH-dependent enzymatic activity of said enzyme is spatially controlled or inhibited or promoted or activate
  • modes of controlling the enzymatically-catalysed formation of said covalent bond comprise particularly inhibition, promotion, activation and / or changing the enzymatic activity of the enzyme, particularly the rate constant of the enzymatic reaction.
  • said enzyme is an aminoacyltransferase (E. C. 2.3.2.), particularly a:transglutaminase (E. C. 2.3. 2. 13), more particular factor XI I la (described by the UniProt Nr: P00488) or precursor thereof,
  • the first moiety is an acyl, particularly an amide
  • the second moiety is an amine.
  • non- limiting examples for such first and second moiety can be taken from Example 7 below.
  • FXIIIa for example uses a free amine group (e.g., protein- or peptide-bound lysine) and the acyl group at the end of the side chain of protein- or peptide-bound glutamine.
  • a free amine group e.g., protein- or peptide-bound lysine
  • the acyl group at the end of the side chain of protein- or peptide-bound glutamine.
  • UniProt numbers refer to entries in the UniProt Knowledgebase.
  • the E. C. number refers to the so-called Enzyme Commission number that is a numerical classification scheme for enzymes.
  • the first and/or second compound consists of or comprises a polymer, wherein said polymer is a natural polymer, particularly one of the following polymers: fibrin, alginate, chitosan, hyaluronic acid, chondroitin sulfate, heparin; or a synthetic polymer, particularly one of the following polymers: polyethylene glycol (PEG), polyactic acid, SU-8; or any polymer consisting of -or including- a combination of monomers, e.g.
  • PEG polyethylene glycol
  • SU-8 any polymer consisting of -or including- a combination of monomers, e.g.
  • dopamine amine-containing groups such as lysine, cathecols, phosphate containing groups, thiol containing groups, alcohol containing groups, active esters and any polymer or dendrimer containing any of said groups (e.g. Hybrane, Boltorn).
  • amine-containing groups such as lysine, cathecols, phosphate containing groups, thiol containing groups, alcohol containing groups, active esters and any polymer or dendrimer containing any of said groups (e.g. Hybrane, Boltorn).
  • the first compound and/ or the second compound comprises polyethylenglycol (PEG), particularly PEG with a molar weight in the range of 4000 to 100000 Dalton, particularly 40000 Dalton, and wherein said PEG is an unbranched or branched PEG and wherein the branched PEG comprises particularly 2, 3, 4, or 8 arms.
  • PEG polyethylenglycol
  • the branched particularly the eight-armed PEG comprises a first or second chemical moiety on particularly each branch or arm of the PEG.
  • the formation of the covalent bond is a condensation reaction, a ligation reaction, a cross-linking reaction or a polymerization reaction.
  • polymerization in the context of the present specification particularly refers to a process of reacting monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks.
  • ligation reaction in the context of the present specification particularly refers to the formation of a covalent bond between two molecules such as amino acids or nucleotides.
  • cross-linking in the context of the present specification particularly refers to the formation of a bond that links one polymer chain to another.
  • condensation reaction in the context of the present specification particularly refers to a chemical reaction in which two molecules or moieties combine to form a larger molecule, together with the loss of a small molecule.
  • small molecules include water, hydrogen chloride, methanol, ammonia, or acetic acid.
  • the voltage is induced via electrodes in said solution, and wherein the voltage is adjusted such that the pH of the solution in proximity of the electrode particularly lies within the range from 1 to 14, particularly 5 to 1 1 .
  • the enzymatic activity of the enzyme is locally inhibited, reduced or promoted depending particularly on the magnitude, polarity of the voltage applied to the solution, and/or the pH of said solution prior to the application of said voltage.
  • the solution comprises a third compound comprising a third moiety, particularly an amide and/or an amine, wherein said third moiety is convertible by said enzyme with the respective first or second moiety, and wherein said third compound is particularly a bioactive molecule, particularly a growth factor, particularly such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), neural growth factor (NGF) and others, or cytokines (e.g. interleukins), affinity linkers (e.g. streptavidin-biotin, ZZ linker, histamine linker), short peptides (e.g. RGD, YIGSR), or proteins and protein fractions (e.g. Laminin, Fibronectin, Fibrinogen, Heparin).
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • NGF neural growth factor
  • cytokines e.g.
  • amide in the context of the present specification particularly refers to a compound comprising an amide (-CONR 2 , wherein each R can independently from the other be hydrogen or an alkyl).
  • amine in the context of the present specification particularly refers to a compound comprising an amine group (-NR 2 , wherein each R can independently from the other be hydrogen or an alkyl).
  • a method for providing a spatially structured hydrogel, particularly using the method according the invention described above, wherein a first compound comprising a first moiety and a second compound comprising a second moiety are linked into a spatially structured hydrogel by formation of a covalent bond between the first and the second moiety of the first and the second compound, and wherein a voltage is applied to a solution comprising the first and the second compound for spatially controlling said formation, wherein said voltage is adjusted such that it induces electrolysis of said solution.
  • the linking reaction particularly the cross- linking reaction a enzymatically catalysed, wherein the first moiety and the second moiety are a substrate of an enzyme, wherein the enzyme is particularly an aminoacyltransferase, particularly a transglutaminase, more particular factor Xllla or a precursor thereof, and wherein the first moiety is particularly an amine and wherein the second moiety is particularly an amide.
  • the pH of the solution is controlled such that the linking reaction is controlled particularly inhibited, promoted, activated or reduced such that the hydrogel exhibits at least in some regions of the hydrogel, particularly on interface regions of the hydrogel, sparsely linked first and second moieties such that particularly an increased cell permeability is achieved.
  • the present method of the local inhibition of the polymerization can be used to create softer interfaces (e.g. less cross-linked polymer) for increased cell permeability.
  • a device for generating a hydrogel particularly using the method according to the invention, wherein said device comprises a reaction chamber that is designed to hold a solution, and a first and a second electrode arranged at the reaction chamber, and particularly a voltage source being designed to apply a pre-defined voltage to said electrodes, so as to induces electrolysis in said solution, particularly so as to control a linking reaction in said solution by locally altering the pH of the solution.
  • the concept of a first and a second electrode may particularly also mean that there is one electrode, wherein the other electrode (e.g. counter electrode) may be formed or comprised by another part of the device such as a wall or bottom of the reaction chamber (or even another electrode). Further, it is also possible to have a plurality of electrodes at which the linking reaction is to be controlled (see above).
  • the first and / or second electrode is designed to be moved non-destructively with respect to the reaction chamber and/ or released from the reaction chamber, particularly so as to remove the first and/ or second electrode from the hydrogel.
  • said electrode is removable from said hydrogel in the reaction chamber without damaging/ straining or destroying said hydrogel.
  • auxiliary components comprising tubing to access the reaction chamber particularly embedded in the electrode structure, membranes to separate the electrodes and a container to separate the hydrogel from the external environment can be comprised by the reaction chamber device according to the invention.
  • the first and/or second electrode comprises a metal, particularly tungsten, a semiconductor, a conductive polymeric material, or a combination of these materials.
  • the first electrode can assume anodic polarization or cathodic polarization, turning it into an anode or a cathode.
  • the second electrode would assume the opposite polarity of the first electrode.
  • the first and/ or second electrode comprises a surface, wherein said surface comprises a region comprising a two- dimensional and/or three-dimensional structure wherein said structure being designed to act as a template for structuring the hydrogel by spatially controlling, particularly inhibiting or promoting, the hydrogel formation, particularly in the proximity of the structure, particularly when said pre-defined voltage is applied to said electrodes.
  • the first and / or second electrode is formed as an elongated element, particularly as a wire, wherein said wire is particularly designed such that if the voltage is applied between the first and second electrode the formation of the hydrogel is inhibited, reduced or promoted in a proximity of the electrodes, wherein if the formation of the hydrogel is inhibited or reduced a hydrogel comprising a hollow channel can be formed, when the electrode is removed from said hydrogel.
  • the first and the second electrode are designed to induce an electrical current density between 0 and ⁇ 100 ⁇ /mm 2 in said solution in the reaction chamber.
  • the first and/or second electrode comprises a cavity and/or a channel that are designed to hold and/or transport a liquid.
  • Such cavities or channels might act as a microfluidic device, where liquids can be handled and flown through for various purposes, but particularly for delivering said liquid to the solution or the formed hydrogel. Furthermore such a liquid might contain biological cells that are introduced via said electrode in the hydrogel particularly after or during formation.
  • the first and / or second electrode is designed as a conductive scanning probe, particularly as an electrochemical atomic force microscope probe or a glass micro-electrode, wherein said conductive scanning probe particularly comprises a scanning tip that is preferably designed such that when a voltage is applied to the conductive scanning probe the electrical field will be highest in the proximity of the scanning tip, and wherein preferably said scanning probe is designed to be moveable, e.g. three- dimensionally movable, with respect to the reaction chamber (or hydrogel residing in the reaction chamber).
  • the scanning probe may be arranged at, in or on the reaction chamber, particularly in an interior of the reaction chamber, and may be removable from the reaction chamber.
  • the scanning probe is movable by means of an actuator such as a known scanning stage, particularly an atomic force microscope stage.
  • a system for generating a spatially structured hydrogel comprising a device according to the invention, wherein the system further comprises said solution for generating a hydrogel and/ or a hydrogel that is placed in said reaction chamber of the device according to the invention.
  • Claim 20 may be directed to a device according to one of the claims 17 to 19 characterized in that the first and/ or second electrode (1 1 , 12) comprises a surface, wherein said surface comprises a structure being designed to act as a template for structuring the hydrogel (200) by spatially controlling, particularly inhibiting or promoting, the hydrogel (200) formation, particularly in the proximity of the first and / or second electrode (1 1 , 12), particularly when said pre-defined voltage is applied to said electrodes (1 1 , 12).
  • Claim 21 may be directed to a device according to one of the claims 17 to 20, characterized in that the first and / or second electrode (1 1 , 12) is formed as an elongated element, particularly as a needle or a wire.
  • Claim 22 may be directed to a device according to one of the claims 17 to 21 , characterized in that the first and the second electrode (1 1 , 12) are designed to induce an electrical current density between 0 and ⁇ 100 ⁇ /mm 2 in said solution in the reaction chamber (100).
  • Claim 23 may be directed to a device according to one of the claims 17 to 22 characterized in that the first and/or second electrode (1 1 , 12) comprises a cavity and/or a channel that are designed to hold and/or transport a liquid.
  • Claim 24 may be directed to a device according to one of the claims 17 to 23 characterized in that the first and / or second electrode (1 1 , 12) is designed as a conductive scanning probe, particularly as an electrochemical atomic force microscope probe or a glass micro-electrode, wherein said conductive scanning probe particularly comprises a scanning tip (121 ), particularly for locally (e.g. namely at the tip) controlling/influencing said solution/formation of said hydrogel as described herein, wherein said tip is particularly designed such that when a voltage is applied to the conductive scanning probe the electrical field will be highest in the proximity of the scanning tip (121 ), and wherein particularly said scanning probe is (e.g. three- dimensionally) movable, particularly in an interior (102) of the reaction chamber (100).
  • a conductive scanning probe particularly as an electrochemical atomic force microscope probe or a glass micro-electrode
  • said conductive scanning probe particularly comprises a scanning tip (121 ), particularly for locally (e.g. namely at the tip) controlling/influencing said solution/
  • Claim 25 may be directed to a system for generating a spatially structured hydrogel comprising a device according to one of the claims 17 to 24, wherein the system further comprises said solution for generating a hydrogel (200) and/or a hydrogel (200) that is placed in said reaction chamber (100) of the device.
  • Fig. 1 shows a perspective view of a device according to the invention
  • Fig. 2 shows a schematic view of a device according to the invention
  • Fig. 3 shows a schematic view of a device according to the invention
  • Fig. 4 shows a schematic view of a device according to the invention
  • Fig. 5 shows a schematic view of a device according to the invention
  • Fig. 6 shows a schematic illustration of the pH-dependency of a cross-linking reaction
  • Fig. 7 shows a schematic illustration of the local pH distribution during
  • Fig. 8 shows microscope images of an example of a hydrogel according to the invention
  • Fig. 9 shows quantitative graphs of derived of Figure 8.
  • Fig. 10 shows microscope images of an example of a hydrogel according to the invention
  • Fig. 1 1 shows microscope images and quantification of hydrogel
  • Fig. 12 shows brightfield microscope images of micro-channel fabrication according to the invention and conventional approach
  • Fig. 13 shows live/dead cell assay microscope image
  • Fig. 14 shows a schematic of a structured 3D two-component hydrogel
  • Fig. 15 shows microscope images of a structured 3D two-component hydrogel according to the invention
  • Fig. 16 shows a cell invasion assay co-manufactured with a hydrogel
  • Fig. 17 shows cell distribution in a two-component hydrogel
  • Fig. 18 shows a schematic view of a device according to the invention.
  • Figure 1 to Figure 5 different embodiments and views of a device for conducting the method according to the invention is depicted.
  • Example 6 the preparation of such a device according to the invention is described.
  • the device features a reaction chamber 100, comprising a chamber wall 101 that surrounds an inside 102 of the reaction chamber 100, a first electrode 1 1 that is designed to be anodically or cathodically polarized by applying a voltage between said first electrode 1 1 and a second electrode 12 arranged at the reaction chamber 100, wherein said second electrode 12 assumes the opposite polarity of the first electrode 1 1 when the method according to the invention is conducted in the reaction chamber 100.
  • the reaction chamber 100 is particularly made of polydimethylsiloxane (PDMS) and is placed on a solid support 103.
  • the inside 102 of the reaction chamber 100 is designed to hold a liquid, particularly a solution comprising precursors for a hydrogel.
  • the reaction chamber 100 is located on top of the solid support 103.
  • the solid support 103 is preferably made of glass and is particularly a coverslip for use on a microscope.
  • said glass substrate 103 is a bottom 104 of the reaction chamber 100.
  • the reaction chamber 100 can be bound to the coverslip 103 by surface plasma activation.
  • the reaction chambers 100 volume in which the hydrogel precursors can be poured is approximately 50 ⁇ _.
  • the first and second electrode 1 1 , 12 are designed as straight wires, particularly Tungsten-wires that run parallel to each other through the reaction chamber 100, particularly having the same distance from the bottom 104 of the reaction chamber 100.
  • Both electrodes 1 1 , 12 particularly extend through the reaction chamber 100 connecting and penetrating the chamber wall 101 on two opposite sides of the reaction chamber 100.
  • the first electrode 1 1 and second electrode 12 preferably have a diameter within the range from 50 ⁇ to 0.5 mm.
  • the reaction chamber 100 can contain two opposite openings 105 in the chamber wall 101 suitable to house a third electrode 13.
  • Said third electrode13 being preferably designed as a wire, preferably like the first and second electrode 1 1 , 12, and extending parallel to the first and second electrode 1 1 , 12, wherein the distance 107 between the opening 106 for receiving the second electrode 12 and the opening 105 receiving the third electrode 13 is preferably 1 mm on each side of the reaction chamber 100. But may also be smaller or larger.
  • Figure 2 shows another embodiment according to the invention.
  • the upper panel shows a side view of the device according to the invention and the lower panel shows a top view of the device according to the invention.
  • the second electrode 12 is arranged closer to and particularly on the bottom 104 of the reaction chamber 100 in this embodiment. Otherwise said embodiment has preferably an analogue architecture as the embodiment depicted in Figure 1 .
  • Figure 3 shows another embodiment according to the invention.
  • the upper panel shows a side view of the device according to the invention and the lower panel shows a top view of the device according to the invention.
  • the embodiment depicted in Figure 3 features the first electrode 1 1 arranged on an upper end of the chamber wall 101 , so that it covers the reaction chamber 100 at least partially. Furthermore the first electrode 1 1 has a rectangular contour. Other aspects of this embodiment are realized analogous to the embodiments shown in Figure 1 and 2.
  • Figure 4 shows a side view of another embodiment according to the invention.
  • the first electrode 1 1 from Figure 3 has a region that comprises indentations, spikes or jags 14 that point toward the inside 102 of the reaction chamber 100.
  • Figure 5 shows another embodiment according to the invention.
  • the upper panel shows a side view of the device according to the invention and the lower panel shows a top view of the device according to the invention.
  • the first electrode 1 1 is hollow and is particularly designed for the use as a microfluidic device. It preferably comprises a microfluidic channel 15 that extends along the inside of such a needle-shaped electrode 1 1 .
  • the first electrode 1 1 preferably penetrates the chamber wall 101 only on one side and extends towards the inside 102 of the reaction chamber 100 parallel to the second electrode 12.
  • the microfluidic channel 15 is designed such that fluids can be delivered to the reaction chamber 100.
  • Figure 18 shows the reaction chamber 100 comprising a first (counter-) electrode 1 1 that is arranged at the wall 101 of the reaction chamber 100 and particularly protrudes into the interior 102 (also denoted as inside herein) of the reaction chamber 100.
  • the second (working-) electrode 12 is designed as a conductive scanning probe, particularly as an electrochemical atomic force microscope probe or a glass micro-electrode, particularly comprising a scanning tip 121 that is designed such that an applied voltage to said scanning probe will yield the largest change of pH of a solution in said reaction chamber 100 in proximity of the tip 121 of the scanning probe or second electrode 12.
  • Said second electrode 12 is particularly three-dimensionally (x,y,z direction) movable within the reaction chamber 100, particularly by means of a controllable scanning stage 122.
  • FIG. 6 shows a first compound comprising a first moiety 20, in this case a branched PEG molecule 20 functionalized with preferably a plurality of lysins 21 (Lys-PEG 20L), a second compound 22 comprising a second moiety 23, in this case also a branched PEG molecule 22 functionalized preferably a plurality of glutamines 23 (Gln-PEG 22G), and a third compound, in this case biomolecules 24, 25 functionalized with either lysine 21 or glutamine 23 (Lys-biomolecule 24L, Gln-biomolecule 25G). Lys-PEG 20L and Gln-PEG 22G are called precursors for the hydrogel 200.
  • Said precursors and the third compound 24L, 25G can be mixed with a
  • transglutaminase that is preferably an activated Factor XIII transglutaminase (FXIIIa) enzyme (EC 2.3.2.13 , UniProt Nr: P00488) that catalyzes the formation of a covalent bond between a free amine group, particularly from a Lysine (Lys) 21 and an amide, particularly from a glutamine (Gin) 23.
  • FXIIIa activated Factor XIII transglutaminase
  • Figure 7 depicts schematically the pH change occurring at the electrode interface upon electrolysis of the solution [19].
  • the pH at the anode 30 decreases as more protons 31 are accumulating in the proximity of the anode 30 and the pH increases at the cathode 32 as more hydroxy-ions 33 accumulate near the cathode 32.
  • the production of Lys-PEG 20L and Gln-PEG 22G precursor is described in detail.
  • Example 1 Formation of a hydrogel under different conditions
  • a local change of pH of the solution as described in Figure 6 can be achieved by the application of an electric current to the solution, via electrodes 1 1 , 12, 13 (as described above), inducing electrolysis of the solution.
  • each electrode 1 1 , 12, 13 The extent of the region around each electrode 1 1 , 12, 13 where a linking, /cross- linking / polymerization of the precursors can be inhibited, confined or promoted, depends on the applied current density, pH and buffer capacity of the precursor solution in proximity of an electrode 1 1 , 12, 13.
  • FITC-tagged Lys substrate (Lys-FITC) is admixed to the solution comprising precursors and the FXIIIa.
  • the solution in the present embodiment is a TRIS-buffer having a pH of 7.6. Confocal fluorescence microscopy images of carefully washed hydrogels 200 demonstrate that the hydrogel 200 formation is inhibited in proximity of the anode
  • Example 10 describes the use of CLSM in more detail.
  • a solution (particularly comprising precursors and FXIIIa and Lys-FITC) is casted in the reaction chamber 100 that comprises two electrodes 1 1 , 12.
  • the electrodes are preferably Tungsten-wires.
  • the anode 30 is placed on the left from each microscope image and the cathode 32 is located right from the respective microscope image.
  • Figure 8 a-e shows confocal fluorescence images of the FITC-tagged hydrogels 200 prepared with a Tris concentration of 50 mM wherein the current density was adjusted between 0 and 8 ⁇ /mm 2 ( Figure 8 a: 0 ⁇ /mm 2 , Figure 8 b: 0.5 ⁇ /mm 2 , Figure 8 c: 2 ⁇ /mm 2 , Figure 8 d: 5 ⁇ /mm 2 and Figure 8 e 8 ⁇ /mm 2 ).
  • Figure 8 f-h shows images of hydrogels prepared with a Tris concentration of 10 mM and increasing current densities adjusted between 0 and 2 ⁇ /mm 2 ( Figure 8 f: 0 ⁇ /mm 2 , Figure 8 g: 0.5 ⁇ /mm 2 , Figure 8 8h: 2 ⁇ /mm 2 ).
  • Example 2 Cross-linking in proximity of an electrode: Another embodiment of the invention is realized by preparing solutions containing the precursors, FXIIIa and fluorescence marker (Lys-FITC) as described above but at pH 5 and pH 1 1 respectively. Under both pH conditions the precursors will not be cross- linked when no electric current is applied to the solution ( Figure 10 a and Figure 10 d), as the FXIIIa activity is inhibited.
  • Lis-FITC fluorescence marker
  • the choice of the solutions' pH is made to be symmetrically distant from the optimum enzymatic activity pH of the FXIIIa.
  • cross-linking region extends equally from the anode 30 and from the cathode 32. However, it has not been checked whether the enzymatic activity of FXIIIa decreases symmetrically at acidic and basic pH.
  • the formation of a hydrogel 200 can thus be confined particularly in proximity of an electrode.
  • the FXIIIa activity can be actively controlled, particularly inhibited or activated by electrochemical modulation of the local pH of the solution.
  • Example 3 Removal of electrodes from the hydrogel manufactured according to the invention and assessment of the mechanical stress on hvdrogels upon Tungsten electrode removal:
  • the ability to spatially control the formation of a hydrogel 200 using Lys-PEG 20L and Gln-PEG 22G as precursors can be used to create spatially defined biological
  • Hydrogels 200 produced using particularly Lys-PEG 20L , Gln- PEG 22G and particularly Lys- or Gin- labeled biomolecules 24L, 25G or markers will be referred to TG-PEG (transglutamination-mediated PEG).
  • TG-PEG transglutamination-mediated PEG
  • the formation of particularly micro-channels in three-dimensional hydrogels 200 is a challenge in the development of in vitro scaffolds. Chrobak and colleagues addressed this challenge by placing a stainless steel microneedle prior to a gel polymerization and by subsequently removing said needle to form a micro-channel [20, 21 ].
  • channels can be created extending through the hydrogel along the electrode wire, without mechanically stressing the hydrogel, when said wire is removed after cross- linking of the hydrogel is complete.
  • the mechanical stress induced to the hydrogel during the Tungsten wire removal is quantified by suspending 20 ⁇ fluorescent micro-particles in the hydrogel precursor solution.
  • Figure 1 1 displays micro-particle displacement caused by removal of the electrode for different current densities.
  • the micro- particles after removal of the electrode can be seen as gray dots and the
  • Figure 1 1 shows the average displacement of the micro-particles after removal of the electrode for Figure 1 1 a-c (The black bar: corresponds to Figure 1 1 a, the gray bar corresponds to Figure 1 1 b and the white boxed bar corresponds to Figure 1 1 c).
  • Figure 12 depicts brightfield microscope images from a TG-PEG hydrogel 200 formed at 0 ⁇ /mm 2 ( Figure 12 a,b). It can be seen that in Figure 12a the micro- channel left by the removed electrode is intact whereas the micro-channel in Figure 12b is disrupted after removal of the electrode. Example 1 1 describes in more detail how the displacement of the hydrogel can be quantified.
  • Example 4 Live/Dead assay on mesenchymal stem cells suspended in the hydrogel during hydrogel and micro-channel manufacturing
  • FIG. 13 shows a Live/Dead assay on mesenchymal stem cells (MSCs) suspended in the hydrogel 200 during channel production with 0 ( Figure 13 a) and 5 ⁇ / ⁇ " ⁇ 2 ( Figure 13 b). No difference in cell viability was observed, as almost no dead cells (white arrows) visible.
  • MSCs mesenchymal stem cells
  • Figure 13 b For the Live/Dead assay, 1 0 6 /ml_ human bone marrow derived MSCs are re-suspended in TG-PEG precursor solution. The solution is cross-linked with ( Figure 13 b) and without (figure 13 a) anodic polarization prior to electrode (Tungsten-wire) removal.
  • the Live/Dead assay (Sigma Aldrich, Switzerland) is carried out according to the manufacturer's protocol. Said assay is based on a fluorescent marker that emits in the red spectral region for dead cells and emits in the green spectral region for live cells.
  • Example 5 Electrochemical control of hydrogel cross-linking can be used for creating complex-structured 3D microenvironments that are locally functionalized:
  • a microenvironment made from TG-PEG can be a hydrogel 200 comprising several proteins, peptides and other biomolecules 24, 25 that support the viability of biological entities, such as e.g. cells.
  • Microenvironments that are particularly hydrogels 200 characterized by a defined architecture and by a controlled spatial distribution of chemical moieties, such as biomolecules 24, 25, that can be used to instruct cells in culture can serve as provisional cell-permissive matrices, which provide graded biological cues, much like natural extra-cellular matrices (ECMs), are of great importance.
  • Figures 14 to 17 show an example of the electrochemical control according to the invention of a hydrogel 200 that cross-linking can be used for the production of complex-structured 3D microenvironments (TG-PEG) that are locally functionalized with biological signals.
  • TG-PEG complex-structured 3D microenvironments
  • a two-component biocompatible microenvironment is manufactured.
  • Figure 14 a-d shows a simplified schemes depicting the production of an engineered microenvironment: FITC and IL-4 (interleukine-4) containing hydrogel precursors at pH 5 are cast in a PDMS reaction chamber and locally cross-linked around a cathode applying -5 ⁇ / ⁇ " ⁇ 2.
  • MSCs were perfused in the channel ( Figure 16 h) and invaded the surrounding environment (Figure 16 i, picture taken at day 7).
  • IL4-sensing HEK cells constitutively expressing mCherry ( Figure 17 k), expresses higher YFP levels (Figure 17 I) in proximity of the IL-4-incorporating gel.
  • a Bright field image is shown to locate the electrode ( Figure 17 m).
  • Figure 17 n Ratio (y-axis) of YFP/mCherry expressing cells as function of the distance (x-axis) from the electrode (values represent means and standard deviation of 3
  • Example 13 describes in more detail how the IL-4 response assay can be performed.
  • a hydrogel 200 according to the invention is sequentially produced: In a first step a cross-linking reaction of a pH 5 precursor solution comprising also FITC and IL-4 functionalized to be convertible by FXIIIa is poured in the reaction chamber 100. A TG-PEG hydrogel 200 forms around the cathode 32 by applying -5 ⁇ /mm 2 ( Figure 14 a).
  • the PDMS reaction chamber 100 is backfilled with another Alexa 561 -Gin-labelled TG-PEG hydrogel precursor solution (pH 7.6) ( Figure 14 c), in which a channel is created as particularly described according to example 3 above ( Figure 14 d).
  • Alexa561 is a fluorescent dye emitting at a different (red-shifted) wavelength than FITC.
  • Various cell types ranging from bone marrow derived MSCs, preosteoblasts or fibroblasts can be delivered to the channel and invade the surrounding environment, that for example contains biomolecules 24, 25 incorporated, particularly growth factors ( Figure 16 i).
  • YFP yellow fluorescent protein
  • Example 6 Preparing a reaction chamber with wire-electrodes according to the invention:
  • Polydimethylsiloxane (PDMS) reaction chambers 200 are made as follows: the silicon elastomer and the curing agent (Sylgard 184, Dow Corning Corporation, USA) are mixed (10:1 in mass) at 2000 rpm for 3 min in a ARE-250 mixer (Thinky Corporation, Japan). The mixture is subsequently poured into poly(methyl methacrylate) (PMMA) moulds, where 500 ⁇ in diameter stainless steel wires are positioned to create admission holes 105, 106 in the wall 101 of the chamber 100 for the electrodes 1 1 , 12, 13. The mixture is subsequently degassed for 30 min in a vacuum chamber and baked for 4 h at 60 °C.
  • PMMA poly(methyl methacrylate)
  • the stainless steel wires and the PDMS reaction chamber 100 are removed from the PMMA moulds, rinsed with isopropanol (IPA), oxygen plasma cleaned (1 min at 300 W, Plasma-System 100, Technics Plasma GmbH, Germany) and finally pressed onto microscope glass cover slips 103.
  • Straightened Tungsten wires 1 1 , 12, 13 (W, 500 ⁇ in diameter, Advent Research Materials Ltd, UK) are inserted in the PDMS reaction chamber 100 and connected to a potentio-galvanostat in a two electrode setup (PGU-10V-1A-IMP-S and ECMwin computer interface, Elektroniklabor Peter Schrems, Germany).
  • Example 7 Production of Lys- Gln-PEG precursors according to the invention: Eight-arm PEG 20, 22 precursors containing the pending Factor XIII activated (FXIIIa) substrate peptides glutamine acceptor substrate (n-PEG-GIn) or lysine donor substrate containing a MMP-sensitive linker (n-PEG-MMP-sensitive-Lys) are produced and characterized as described elsewhere [4].
  • Eight-arm PEG mol. wt. 40000 is purchased from Nektar (Huntsville, AL, USA). Divinyl sulfone is purchased from Aldrich (Buchs, Switzerland). PEG vinylsulfone (PEG-VS) is produced and characterized as described elsewhere [22].
  • the FXIIIa substrate peptides H-NQEQVSPL-ERCG-NH2 (TG-GIn) Ac-FKGG-GPQGIWGQ-ERCG-NH2 (MMP-sensitive-Lys), and the adhesion ligand Ac-GCYGRDGSPG-NH2 (TG-Gln- RGD) are obtained from NeoMPS (Strasbourg, France) (immunograde, C18-purified, HPLC analysis: > 90%).
  • the NQEQVSPL cassette corresponds to the FXIIIa substrate site in a2-plasmin inhibitor [23], the FKGG cassette to an optimized FXIIIa substrate site [24], and the ERCG cassette to the vinylsulfone-reactive Cysteine [25].
  • TG-MMP-sensitive-Lys are added to PEG-VS in 1.2-fold molar excess over VS groups and allowed to react in 0.3 M triethanolamine (pH 8.0) at 37°C for 2 h.
  • the products are dialyzed (Snake Skin, MWCO 10K, PIERCE, Rockford, IL, USA) against ultrapure water for 3 days at 4°C. After dialysis, the salt- free products (8-PEG-MMP-sensitive-Lys and 8-PEG-Gln, respectively) are lyophilized.
  • FXIIIa 200 U/mL, Fibrogammin, CSL Behring, Switzerland
  • 100 ⁇ _ of thrombin (20 U/mL, Sigma-Aldrich, Switzerland) for 30 min at 37°C.
  • Small aliquots of activated FXIIIa can be stored at -80°C for further use.
  • Lys-FITC, TG-Alexa 561 , Gln-RGD or combinations are added to the precursor solution prior to initiation of cross-linking by 10 U/mL thrombin-activated FXIIIa and vigorous mixing.
  • Example 9 Electrochemical control of TG-PEG cross-linking according to the invention: To study the effect of electrochemistry on TG-PEG polymerization a 60 ⁇ _ solution composed of TG-PEG, Tris (50 mM or 10 mM, pH 5, 7.6 or 1 1 ), CaCI 2 and a fluorescent agent such as for example Lys-FITC, TG-Alexa 561 or fluorescent polystyrene beads (Fluoresbrite Plain YG 20 micron microspheres, Polyscience Inc.) are mixed with the FXIIIa. The mixture has to immediately be poured in the PDMS reaction chamber. The cross-linking of the TG-PEG is allowed to progress during 6 minutes in presence of a DC current applied in galvanostatic mode. The current density can be varied in a range between 0 and 8 ⁇ /mm 2 .
  • Example 10 Confocal Laser Scanning Microscopy (CLSM):
  • the TG-PEG hydrogel-electrode interfaces are imaged using a LSM 510 confocal laser scanning microscope (Carl Zeiss AG, Germany). It might be necessary to adjust the focal plane to obtain the maximal section of the Tungsten wire (500 ⁇ ).
  • the FITC is detected upon excitation at 490nm with 0.7% laser power, and an emission band pass filter 505-550 nm. Alexa 561 can be detected upon excitation at 515 nm and with an emission band pass filter 575-615 nm.
  • the intensity profiles can be obtained over a 500 x 500 ⁇ field of view by setting the minimum intensity as the average intensity of the electrode and by normalizing the values over the average intensity of the distal 200 ⁇ (maximal intensity). At least 3 samples per condition should be analyzed and 2 images per electrode are acquired.
  • Example 1 1 Quantification of hydrogel displacement upon electrode removal:
  • the displacement of 20 ⁇ polystyrene particles dispersed in the hydrogel 200 is tracked using a Leica fluorescence microscope (BM550B, Leica Microsystems, Germany).
  • the TG-PEG hydrogel 200 is prepared as described above and the Tungsten wires 1 1 , 12, 13 are manually pulled out of the hydrogel 1 1 , 12, 13. Images are recorded every 100 ms and the particles are detected upon excitation at 488 nm. The particle trajectories are calculated using an Image J script previously described [26].
  • Hydrogels were subsequently placed in medium supplemented with human platelet-derived growth factor BB (PDGF-BB, 10 ng/ml, Peprotech, cat. no. 100-14B) and kept in culture for 7 days. Bright field images were acquired with a ZEISS Axiovert 200M inverted microscope.
  • HEK-IL4 reporter cells are produced as described previously [6].
  • HEK 293T cells are transfected with pHW40 (PSTAT6-eYFP-pA) and the constitutive expression vector STAT6 (obtained from Open Biosystems, Huntsville, AL, Clone ID 5530399).
  • a constitutive mCherry expression plasmid (pMK47) is used as internal control.
  • 106/ mL reporter cells are resuspended in TG-PEG precursor solution and cultured for 24 hours in DMEM/F-12 + GlutaMAXTM supplemented with 10% (v/v) fetal calf serum (FCS, Gibco Life Technologies, cat. no.
  • Fluorescent and brightfield images are acquired with a
  • the ratio of cells expressing YFP over cells expressing mCherry might be determined with ImageJ.
  • the IL-4 response in 600 ⁇ wide regions is measured, and the mean and standard deviation calculated out of 3 independent experiments.

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Abstract

L'invention se rapporte à un procédé, à un dispositif et à un système permettant de produire en particulier un hydrogel (200) et de commander une formation par catalyse enzymatique d'une liaison covalente dans une solution, ladite liaison covalente étant formée entre un premier composé (20) comprenant une première fraction (21) et un second composé (22) comprenant une seconde fraction (23), la première (21) et la seconde fraction (23) étant un substrat d'une enzyme, ladite enzyme catalysant la formation d'une liaison covalente entre la première (21) et la seconde fraction (23) et une tension étant appliquée à la solution pour commander dans l'espace ladite formation, ladite tension étant ajustée de telle sorte qu'elle induise une électrolyse de ladite solution.
PCT/EP2014/070820 2013-09-29 2014-09-29 Procédé, dispositif et système permettant de commander électrochimiquement dans l'espace la formation d'un hydrogel WO2015044426A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020008038A1 (en) * 1998-06-24 2002-01-24 Therasense, Inc. Combinatorial Electrochemical Synthesis
US20110171239A1 (en) * 2008-09-26 2011-07-14 Trustees Of Tufts College pH INDUCED SILK GELS AND USES THEREOF
US20130243693A1 (en) * 2010-06-17 2013-09-19 Tufts University Silk optical particles and uses thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2441414T3 (es) * 2002-09-26 2014-02-04 Ajinomoto Co., Inc. Preparación enzimática y procedimiento para producir alimentos que utiliza la misma
US8852620B2 (en) * 2007-07-20 2014-10-07 Medtronic Vascular, Inc. Medical devices comprising polymeric drug delivery systems with drug solubility gradients
JP5885505B2 (ja) * 2008-09-26 2016-03-15 タフツ ユニバーシティー／トラスティーズ オブ タフツ カレッジ 活性なシルク粘液接着剤、シルク電気的ゲル化方法、およびデバイス
CA2832795C (fr) * 2010-04-12 2018-01-09 Tufts University Composants electroniques en soie et la methode de fabrication desdits composants

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020008038A1 (en) * 1998-06-24 2002-01-24 Therasense, Inc. Combinatorial Electrochemical Synthesis
US20110171239A1 (en) * 2008-09-26 2011-07-14 Trustees Of Tufts College pH INDUCED SILK GELS AND USES THEREOF
US20130243693A1 (en) * 2010-06-17 2013-09-19 Tufts University Silk optical particles and uses thereof

Non-Patent Citations (34)

* Cited by examiner, † Cited by third party
Title
A. M. KLOXIN; A. M. KASKO; C. N. SALINAS; K. S. ANSETH, SCIENCE, vol. 324, 2009, pages 59
A. SALA; P. HANSELER; A. RANGA; M. P. LUTOLF; J. VOROS; M. EHRBAR; F. E. WEBER, INTEGRATIVE BIOLOGY, vol. 3, 2011, pages 1102
B. DERBY, SCIENCE, vol. 338, 2012, pages 921
B. W. MAYNOR; S. F. FILOCAMO; M. W. GRINSTAFF; J. LIU, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 124, 2001, pages 522
B.-H. HU; P. B. MESSERSMITH, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 125, 2003, pages 14298
C. A. DEFOREST; B. D. POLIZZOTTI; K. S. ANSETH, NAT MATER, vol. 8, 2009, pages 659
C. LASLAU; D. E. WILLIAMS; B. KANNAN; J. TRAVAS-SEJDIC, ADVANCED FUNCTIONAL MATERIALS, vol. 21, 2011, pages 4607
D. BHATTACHARJYA; I. MUKHOPADHYAY, LANGMUIR, vol. 28, 2012, pages 5893
E. DE GIGLIO; S. COMETA; C. SATRIANO; L. SABBATINI; P. G. ZAMBONIN, JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A, vol. 88A, 2009, pages 1048
I. F. SBALZARINI; P. KOUMOUTSAKOS, JOURNAL OF STRUCTURAL BIOLOGY, vol. 151, 2005, pages 182
J. A. BURDICK; A. KHADEMHOSSEINI; R. LANGER, LANGMUIR, vol. 20, 2004, pages 5153
J. C. SCHENSE; J. A. HUBBELL, BIOCONJUGATE CHEMISTRY, vol. 10, 1998, pages 75
J. S. MILLER; C. J. SHEN; W. R. LEGANT; J. D. BARANSKI; B. L. BLAKELY; C. S. CHEN, BIOMATERIALS, vol. 31, 2010, pages 3736
J. S. MILLER; K. R. STEVENS; M. T. YANG; B. M. BAKER; D.-H. T. NGUYEN; D. M. COHEN; E. TORO; A. A. CHEN; P. A. GALIE; X. YU, NAT MATER, vol. 11, 2012, pages 768
J. T. KIM; S. K. SEOL; J. PYO; J. S. LEE; J. H. JE; G. MARGARITONDO, ADVANCED MATERIALS, vol. 23, 2011, pages 1968
J. UQUILLAS; O. AKKUS, ANN BIOMED ENG, vol. 40, 2012, pages 1641
K. M. CHROBAK; D. R. POTTER; J. TIEN, MICROVASCULAR RESEARCH, vol. 71, 2006, pages 185
M. CORDEY; M. LIMACHER; S. KOBEL; V. TAYLOR; M. P. LUTOLF, STEM CELLS, vol. 26, 2008, pages 2586
M. EHRBAR; A. SALA; P. LIENEMANN; A. RANGA; K. MOSIEWICZ; A. BITTERMANN; S. C. RIZZI; F. E. WEBER; M. P. LUTOLF, BIOPHYSICAL JOURNAL, vol. 100, 2011, pages 284
M. EHRBAR; R. SCHOENMAKERS; E. H. CHRISTEN; M. FUSSENEGGER; W. WEBER, NAT MATER, vol. 7, 2008, pages 800
M. EHRBAR; S. C. RIZZI; R. G. SCHOENMAKERS; B. SAN MIGUEL; J. A. HUBBELL; F. E. WEBER; M. P. LUTOLF, BIOMACROMOLECULES, vol. 8, 2007, pages 3000
M. EHRBAR; S. C. RIZZI; R. HLUSHCHUK; V. DJONOV; A. H. ZISCH; J. A. HUBBELL; F. E. WEBER; M. P. LUTOLF, BIOMATERIALS, vol. 28, 2007, pages 3856
M. GABI; T. SANNOMIYA; A. LARMAGNAC; M. PUTTASWAMY; J. VOROS, INTEGRATIVE BIOLOGY, vol. 1, 2009, pages 108
M. P. LUTOLF; J. A. HUBBELL, BIOMACROMOLECULES, vol. 4, 2003, pages 713
M. P. LUTOLF; J. A. HUBBELL, NAT BIOTECH, vol. 23, 2005, pages 47
M. P. LUTOLF; N. TIRELLI; S. CERRITELLI; L. CAVALLI; J. A. HUBBELL, BIOCONJUGATE CHEMISTRY, vol. 12, 2001, pages 1051
M. V. TSURKAN; K. CHWALEK; S. PROKOPH; A. ZIERIS; K. R. LEVENTAL; U. FREUDENBERG; C. WERNER, ADVANCED MATERIALS, vol. 25, 2013, pages 2606
N. TIRELLI; M. P. LUTOLF; A. NAPOLI; J. A. HUBBELL, REVIEWS IN MOLECULAR BIOTECHNOLOGY, vol. 90, 2002, pages 3
N. ZAARI; P. RAJAGOPALAN; S. K. KIM; A. J. ENGLER; J. Y. WONG, ADVANCED MATERIALS, vol. 16, 2004, pages 2133
P. S. LIENEMANN; M. KARLSSON; A. SALA; H. M. WISCHHUSEN; F. E. WEBER; R. ZIMMERMANN; W. WEBER; M. P. LUTOLF; M. EHRBAR, ADVANCED HEALTHCARE MATERIALS, vol. 2, 2013, pages 292
R. G. WYLIE; S. AHSAN; Y. AIZAWA; K. L. MAXWELL; C. M. MORSHEAD; M. S. SHOICHET, NAT MATER, vol. 10, 2011, pages 799
S. JEGADESAN; R. C. ADVINCULA; S. VALIYAVEETTIL, ADVANCED MATERIALS, vol. 17, 2005, pages 1282
S. K. SEOL; J. T. KIM; J. H. JE; Y. HWU; G. MARGARITONDO, MACROMOLECULES, vol. 41, 2008, pages 3071
SANBORN T J ET AL: "In situ crosslinking of a biomimetic peptide-PEG hydrogel via thermally triggered activation of factor XIII", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 23, no. 13, 1 July 2002 (2002-07-01), pages 2703 - 2710, XP004352578, ISSN: 0142-9612, DOI: 10.1016/S0142-9612(02)00002-9 *

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